Joyce’s Weblog

BioMinds Research Day se llevo a cabo el 21 de Marzo de 2009 en el Centro de Bioprocesos de Mayaguez. Durante este dia tuvimos la oportunidad de reunirnos como grupo para escuchar conferencias y presentar nuestros poster. Luego de trabajar durante un año y medio, presentamos nuestros proyectos de una manera coherente. La interaccion directa con otros estudiantes interesados en la investigacion fue de gran aprendizaje.

Este dia tuve la oportunidad de evaluar a N. Melendez, J.M. Caraballo y E.C. Gutierrez, todos parte del programa.

N. Melendez. FT-IR Spectra and 2D-COS Analysis for the Thermal Perturbation of hCenz.

Fue un poster interesante y facil de seguir (a pesar que no es mi area de estudio). La Srta. Melendez mostro gran conocimiento de su data y de su tema. Su estudio es de gran relevancia ya que la segregacion de los cromosomas durante mitosis es esencial para la celula.

J.M. Caraballo. Synthesis and characterization of the Platinum Complexes

La relevancia de su investigacion es crear nuevos tratamientos para cancer. Durante la misma tuve la preocupacion de que la molecula que estan desarrollando contiene platino. Caraballo pudo explicarme claramente su uso y ventajas.

E.C. Gutierrez. Pathway to a Synapse: Motor Protein Mediated Transport of Cell Adhesion Molecules at the Onset of Synaptogenesis.

Encontre que su abstract proveyo un resumen completo de su trabajo y su poster estuvo bien organizado. Gutierrez no mostro nervios durante la presentacion y su seguridad y conocimiento del trabajo fue admirable.

Hacer investigacion es toda una experiencia que todos deberian experimentar. Es enfrentarse a diferentes problemas y resolverlos de la mejor manera posible. En el camino se encuentran muchas dificultades pero es gratificante poder sobrepasarlas…

An Interaction between Mex67p and Upf1p at the Terminal Step

of mRNA Export Facilitates mRNA Translation

Abby J. Agosto, Iván J. Cajigas, Paloma M. Guzzardo, Heike Krebber and

Carlos I. González

Gene expression relies on the communication between the processes of transcription, mRNA processing, export and translation. Once synthesized in the nucleus, an mRNA must become competent for its translocation across the nuclear pore. Formation of an mRNA particle (mRNP) suitable for export is accomplished by the binding of soluble export factors to the nascent mRNA. Following transport, mRNPs must undergo structural rearrangements in the cytoplasm in order to enter translation. We have gathered information to support this mRNP remodeling step. Specifically, we have found that Mex67p interacts with Upf1p. We provide evidence to support the notion that the Mex67p-Upf1p interaction is neither required for the activation of the NMD pathway or for poly-(A)+ mRNA export. Instead, Upf1p and Mex67p are critical to facilitate the conversion from an export-competent mRNP to a translation-competent mRNP, since cells lacking both proteins demonstrate severe defects in protein synthesis. Specifically, our data demonstrate that these cells are unable to efficiently initiate mRNA translation and recognize translation termination codons. We propose that in the cytoplasm, Upf1p communicates the Mex67p-bound mRNAs with the protein synthesis machinery to facilitate translational competency and couple the terminal step of mRNA export to mRNA translation.

Since cells harboring the mutant version of Mex67 (export factor) and lacking Upf1 (NMD factor) demonstrate high molecular weight aggregates, we are directed towards determining if Mex67p can exist in a prion like form. In order to further characterize this behavior we will verify the Q/N content of the protein and look for phenotypes that can be reversed in the presence of Guanidine HCl (GuHCl) which will indicate possible prion disruption.

Experimental Design:

      Using yeast genetics, we will verify phenotypes that can be used as markers for prion disruption in the presence of GuHCl. These phenotypes may include:

1.  
   1. temperature sensitivity
   2. drug sensitivity (cycloheximide or paromomicin)
   3. colony size

      Additional assays for scoring the hability of mex67-5 to form prions is the cytoduction experiment. These experiments will determine if the molecular weight aggregates have a non mendelian inheritance pattern tipical of prion proteins.

Un semestre que casi acaba en tan solo un instante. Durante el inicio del semestre tuve la oportunidad de trabajar en el bench con más frecuencia de lo que lo hice al final del semestre. Realicé curvas de crecimiento en triplicado a 24, 30 y 33°C. Con estos datos confirmé data anterior acerca de los defectos en crecimiento que muestra la mutante doble del factor de exporte, Mex67, y el factor envuelto en NMD, Upf1. Concluimos que el efecto en crecimiento se presenta a los 30 y 33°C, reconfirmando y reforzando la data anterior. Utilizando el mismo concepto de crecimiento de levaduras, pero utilizando la técnica de diluciones en serie, determinamos el efecto de añadir la droga Canavinina, lo que sugiere defectos en terminación de traducción ya que nuestras cepas poseen el alelo can1-100. Obtuvimos que para la doble mutante (mex67-5, upf1∆) los defectos en crecimiento en presencia de esta droga son más pronunciados a 33°C.

Al iniciar el semestre planeaba hacer curvas de crecimiento en diferentes condiciones y polysome profiles y aún tener tiempo suficiente para trabajar más a fondo. Sin embargo, no fue posible realizar los polysome profiles por dificultades técnicas, pero se vió el efecto de Canavinina (lo cual no estaba planeado). Mi trabajo finalizando este semestre ha sido de buscar en la literatura para identificar diferentes maneras de caracterizar más a fondo la interacción entre los factores de interés.

Tengo muchos planes diversos para el próximo semestre, los cuales trabajaré y definiré en cuanto vayan tomando forma. Entre las cosas que deseo realizar el próximo son trabajar los polysome profiles y determinar si nuestra doble mutante presenta defectos en iniciación de traducción, entre otras cosas.

Mi trabajo contribuye al área de las Ciencias Biológicas porque se enfoca en describir el mecanismo de acción que una célula eucariótica posee para detectar mensajeros “saludables” y traducir proteínas funcionales. Esto es necesario ya que el código genético debe ser expresado y traducido correctamente para sintetizar polipéptidos funcionales que aseguren una célula, un tejido, un órgano o un organismo saludable.

Es relevante conocer y caracterizar proteinas envueltas en exporte y traduccion de mensajero por la aplicacion Biomedica. Ciertas enfermedades como la distrofia muscular se deben a codones de terminacion prematuros lo que conlleva la sintesis de una proteina truncada. Upf1 esta envuelta en el proceso de reconocimiento y degradacion de los mRNA con esos codones prematuros. En adicion, se poseen otros mecanismos para asegurar la expresion de proteinas funcionales. La interaccion entre Mex67 y Upf1 que estudiamos en mi laboratorio podria ser parte de un mecanismo celular de reconocimiento de mensajeros “saludables”.

He avanzado en mi proyecto, sin embargo, estoy en un momento de buscar en la literatura.

Encuentro que he hecho gran avance en cuanto a los planes establecidos al inicio del semestre; lo clasifico como un 3. Entiendo que estoy bastante avanzada en el proyecto dado que solo va un mes desde que comence a trabajar. Bueno, al menos he progresado en la primera mitad y encuentro que la parte que me queda (a pesar de ser solamente dos experimentos) me tomaran el resto del semestre debido a la planeacion y el tiempo que requieren los “polysome profiles”.

Con respecto a mis metas, todo se ha movido segun planeado (si no tomo en cuenta que las curvas de crecimiento requieren 17 horas corridas, todo a estado bajo control). Para afrontar el problema de estar muchas horas en el laboratorio, he decidido trabajar los fines de semana y… mucho cafe.

This summer, I was given the opportunity to do research in Dr. Joshua Rabinowitz laboratory at Princeton University. It was a great experience. I was working with a different area of Science (Biochemistry) and a different model organism (E. coli). I learned many new techniques and it presented me to a new way of analyzing the data, which helps me expand my knowledge as a scientist. It was very challenging at the beginning but with effort and dedication I finished the summer more than satisfied with my work.

During this semester, at Dr. Carlos I. Gonzalez laboratory, I expect to further characterize the role of Mex67 and Upf1 in translation. In order to accomplish this, I plan to make growth curves in different conditions and at different temperatures to view growth defects. Also, I expect to perform many polysome profiles followed by Western Blots at different temperatures in order to confirm my previous data and to have an idea of the molecular behavior of my strains.

As you may notice, my project is directed, since last semester, toward the elucidation of the role of Mex67 and Upf1 in translation, which is my main topic. As a new technique, I plan to make some growth curves in order to measure quantitatively the growth defects that we have noticed before.

I have learned that it is necessary to read the literature and look for other ways of thinking that are not necessarily the conventional ones. I am looking to find great experiences that will help me develop as an integral professional. I want to become a scientist and make my family proud of my accomplishments. I want to gain more research experience, learn new ways of approaching investigation and broaden my knowledge in Molecular Biology and Biochemistry.

The biggest challenge during this semester has been doing the experiments by myself. This is so, because it is difficult to work independently, also developing short-term plans is a hard work that requires a high level of organization and perseverance.

For the next semester, I’m expecting to do well while working without my graduate mentor, who graduates this semester.  I wish to elucidate the role of Mex67 and the NMD factor (Upf1) in translation, as well as characterize other factors involved.

During this semester in the lab, I have dealed with certain challenges presented during the execution of the experiments. Even so, it has been extremely fascinating and satisfactory. During this time, I have mastered the techniques that I have already learned. In addition, I have learned to perform Polysome Profiles and purify proteins from sucrose gradients. This is relevant in the development of my project because they allow us to explore mRNA translation defects.

The networking made using the Bio Blog has been great since I have been able to learn about different experiments and techniques that undergraduate students are doing. It is interesting because these are projects running in Puerto Rico, showing that this country has a great amount of students  devoted with their careers.

During my second semester at the University of Puerto Rico, I was given the opportunity to work at Dr. Carlos I. González laboratory. His laboratory is interested in the control of gene expression at the post transcriptional level in eukaryotic cells. I am specifically working with two different projects in the yeast Saccharomyces cerevisiae. Both research projects are related to the Nonsense-mediated mRNA decay (NMD) pathway, an mRNA surveillance mechanism that is responsible for the rapid turnover of mRNAs harboring premature termination codons. My first project focuses on determining the role of an interaction between a cis-acting element (Downstream Sequence Element or DSE) in the RNA and a nucleocytoplasmic shuttling protein (Hrp1p), and how this interaction promotes the degradation of aberrant messenger RNAs. The second project is directed towards the identification of the role of Mex67p and Upf1p in translation termination. Along with the findings that we already made, came a new question of how an export protein could be involved in translation termination. In order to test this possibility, we have decided to characterize Mip6p, a protein that interacts with the termination factor (Sup35) and Mex67p. To be able to carry out these projects I have learned techniques such as: Northern Blots, Dual Luciferase Assays, PCRs, in vitro transcription, SDS-PAGE, gel electrophoresis, among others. I have learned that it is necessary to read the literature and look for other ways of thinking that are not necessarily the conventional ones. As a college student, I am looking to find great experiences that will help me develop as an integral professional. I want to become a scientist and make my family proud of my accomplishments. I want to gain more research experience, learn new ways of approaching investigation and broaden my knowledge in Molecular Biology and Biochemistry.